The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Many modern advances in cellular and molecular biology are rooted in the advent of large-scale amplification of nucleic acids and analytical methods dependent thereon. A number of methods are known in the art for performing such amplification of template nucleic acid molecules to generate populations of substantially identical copies. One technique that is particularly amenable to high throughput applications is emulsion polymerase chain reaction (“emulsion PCR” or “emPCR”).
Emulsion PCR is performed by isolation of individual DNA molecules along with primer-coated beads in aqueous droplets within an oil phase. A PCR step coats each bead with clonal copies of the DNA molecule which are then immobilized for later sequencing. Emulsion PCR is used in a number of commercial methods, such as those of 454 Life Sciences, and SOLiD sequencing, (developed by Agencourt, now Applied Biosystems). Current emulsion PCR techniques involve the use of small fragments of DNA, which renders it unsuitable for analysis of certain genotypes such as those depending on an allelic linkage, and other applications for which assessment of large nucleic acids are required. Therefore, a need remains for emulsion PCR-based analytical methods that may applied to the evaluation of large nucleic acids including, for example, for monosomal analysis.